The biological activation of N2 occurs at the FeMo‐cofactor, a 7Fe–9S–Mo–C–homocitrate cluster. FeMo‐cofactor formation involves assembly of a Fe6–8–SX–C core precursor, NifB‐co, which occurs on the ...NifB protein. Characterization of NifB‐co in NifB is complicated by the dynamic nature of the assembly process and the presence of a permanent 4Fe–4S cluster associated with the radical SAM chemistry for generating the central carbide. We have used the physiological carrier protein, NifX, which has been proposed to bind NifB‐co and deliver it to the NifEN protein, upon which FeMo‐cofactor assembly is ultimately completed. Preparation of NifX in a fully NifB‐co‐loaded form provided an opportunity for Mössbauer analysis of NifB‐co. The results indicate that NifB‐co is a diamagnetic (S=0) 8‐Fe cluster, containing two spectroscopically distinct Fe sites that appear in a 3:1 ratio. DFT analysis of the 57Fe electric hyperfine interactions deduced from the Mössbauer analysis suggests that NifB‐co is either a 4Fe2+–4Fe3+ or 6Fe2+–2Fe3+ cluster having valence‐delocalized states.
Clearing up an Fe‐cluster fluster: The biological activation of N2 occurs at the FeMo‐cofactor, a 7Fe–9S–Mo–C–homocitrate cluster. FeMo‐cofactor formation involves assembly of a (6‐8)Fe–xS–C core precursor, NifB‐co, which occurs on the NifB protein. Mössbauer analysis on an in vivo purified NifB‐co bound to NifX, a physiological carrier protein, indicates that NifB‐co is a diamagnetic (S=0) 8‐Fe cluster, which contains two spectroscopically distinct Fe sites in a 3:1 ratio.
Surprisingly uninhibited: The inhibition of hydrogenases by oxygen is intensely studied because this is the main obstacle to using these enzymes in biofuel cells. The hydrogenase from Clostridium ...acetobutylicum (see structure) was found to react surprisingly slowly with O2. The inhibition mechanism was elucidated and the kinetics were quantitatively defined. This is a prerequisite for improving the enzyme further by genetic engineering and for assessing its potential in technological devices.
The establishment of polarity is a critical process in pathogenic fungi, mediating infection-related morphogenesis and host tissue invasion. Here, we report the identification of TPC1 (Transcription ...factor for Polarity Control 1), which regulates invasive polarized growth in the rice blast fungus Magnaporthe oryzae. TPC1 encodes a putative transcription factor of the fungal Zn(II).sub.2 Cys.sub.6 family, exclusive to filamentous fungi. Tpc1-deficient mutants show severe defects in conidiogenesis, infection-associated autophagy, glycogen and lipid metabolism, and plant tissue colonisation. By tracking actin-binding proteins, septin-5 and autophagosome components, we show that Tpc1 regulates cytoskeletal dynamics and infection-associated autophagy during appressorium-mediated plant penetration. We found that Tpc1 interacts with Mst12 and modulates its DNA-binding activity, while Tpc1 nuclear localisation also depends on the MAP kinase Pmk1, consistent with the involvement of Tpc1 in this signalling pathway, which is critical for appressorium development. Importantly, Tpc1 directly regulates NOXD expression, the p22.sup.phox subunit of the fungal NADPH oxidase complex via an interaction with Mst12. Tpc1 therefore controls spatial and temporal regulation of cortical F-actin through regulation of the NADPH oxidase complex during appressorium re-polarisation. Consequently, Tpc1 is a core developmental regulator in filamentous fungi, linking the regulated synthesis of reactive oxygen species and the Pmk1 pathway, with polarity control during host invasion.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Generation of mRNA isoforms by alternative polyadenylation (APA) and their involvement in regulation of fungal cellular processes, including virulence, remains elusive. Here, we investigated ...genome-wide polyadenylation site (PAS) selection in the rice blast fungus to understand how APA regulates pathogenicity. More than half of Magnaporthe oryzae transcripts undergo APA and show novel motifs in their PAS region. Transcripts with shorter 3'UTRs are more stable and abundant in polysomal fractions, suggesting they are being translated more efficiently. Importantly, rice colonization increases the use of distal PASs of pathogenicity genes, especially those participating in signalling pathways like 14-3-3B, whose long 3'UTR is required for infection. Cleavage factor I (CFI) Rbp35 regulates expression and distal PAS selection of virulence and signalling-associated genes, tRNAs and transposable elements, pointing its potential to drive genomic rearrangements and pathogen evolution. We propose a noncanonical PAS selection mechanism for Rbp35 that recognizes UGUAH, unlike humans, without CFI25. Our results showed that APA controls turnover and translation of transcripts involved in fungal growth and environmental adaptation. Furthermore, these data provide useful information for enhancing genome annotations and for cross-species comparisons of PASs and PAS usage within the fungal kingdom and the tree of life.
NifB-co, an Fe−S cluster produced by the enzyme NifB, is an intermediate on the biosynthetic pathway to the iron molybdenum cofactor (FeMo-co) of nitrogenase. We have used Fe K-edge extended X-ray ...absorption fine structure (EXAFS) spectroscopy together with 57Fe nuclear resonance vibrational spectroscopy (NRVS) to probe the structure of NifB-co while bound to the NifX protein from Azotobacter vinelandii. The spectra have been interpreted in part by comparison with data for the completed FeMo-co attached to the NafY carrier protein: the NafY:FeMo-co complex. EXAFS analysis of the NifX:NifB-co complex yields an average Fe−S distance of 2.26 Å and average Fe−Fe distances of 2.66 and 3.74 Å. Search profile analyses reveal the presence of a single Fe−X (X = C, N, or O) interaction at 2.04 Å, compared to a 2.00 Å Fe−X interaction found in the NafY:FeMo-co EXAFS. This suggests that the interstitial light atom (X) proposed to be present in FeMo-co has already inserted at the NifB-co stage of biosynthesis. The NRVS exhibits strong bands from Fe−S stretching modes peaking around 270, 315, 385, and 408 cm−1. Additional intensity at ∼185–200 cm−1 is interpreted as a set of cluster “breathing” modes similar to those seen for the FeMo-cofactor. The strength and location of these modes also suggest that the FeMo-co interstitial light atom seen in the crystal structure is already in place in NifB-co. Both the EXAFS and NRVS data for NifX:NifB-co are best simulated using a Fe6S9X trigonal prism structure analogous to the 6Fe core of FeMo-co, although a 7Fe structure made by capping one trigonal 3S terminus with Fe cannot be ruled out. The results are consistent with the conclusion that the interstitial light atom is already present at an early stage in FeMo-co biosynthesis prior to the incorporation of Mo and R-homocitrate.
The influence of Fe-hydrogenase from
Clostridium acetobutylicum was studied on the anaerobic corrosion of mild steel. Two short-circuited mild steel electrodes were exposed to the same solution and ...hydrogenase was retained on the surface of only one electrode thanks to a dialysis membrane. The galvanic current and the electrode potential were measured as a function of time in order to monitor the difference in electrochemical behaviour induced by the presence of hydrogenase. A sharp potential decrease of around 500
mV was controlled by the deoxygenating phase. When hydrogenase was introduced after complete deoxygenation, significant heterogeneous corrosion was observed under the vivianite deposit on the electrode in contact with hydrogenase, while the other electrode only showed the vivianite deposit, which was analysed by MEB and EDX. The effect of hydrogenase was then confirmed by monitoring the free potential of single coupons exposed or not to the enzyme in a classical cell after complete deoxygenating. In both phosphate and Tris–HCl buffers, the presence of hydrogenase increased the free potential around 60
mV and induced marked general corrosion. It was concluded that Fe-hydrogenase acts in the absence of any final electron acceptor by catalysing direct proton reduction on the mild steel surface.
The addition of laccase enzymes reduces the
amount of phenols present in lignocellulosic pretreated
materials and increases their fermentability. However,
laccase addition in combination with ...cellulases reduces
hydrolysis yields. In this work, hybrid hydrolysis and
fermentation (HHF) configuration allowed overcoming
the negative effect of laccase treatment on enzymatic
hydrolysis. Furthermore, the effects of different laccase
dosages, length of detoxification time and inoculum size
on ethanol production were evaluated. In the evaluated
configurations, the different laccase dosages did not show
any significant effect on enzymatic hydrolysis. The lowest
laccase dosage (0.5 IU/g DW) removed ~70% of total
phenols which was enough to reach the highest ethanol
production yields (~10 g/L) using K. marxianus CECT
10875. Shorter detoxification times and larger inoculum
sizes had a positive impact on both ethanol production
and volumetric productivity. These optimal detoxification
conditions enable the fermentation of inhibitory slurries by
reducing the overall time and cost of the process.
NafY participates in the final steps of nitrogenase maturation, having a dual role as iron-molybdenum cofactor (FeMo-co) carrier and as chaperone to the FeMo-co-deficient apo-NifDK ...(apo-dinitrogenase). NafY contains an N-terminal domain of unknown function (n-NafY) and a C-terminal domain (core-NafY) necessary for FeMo-co binding. We show here that n-NafY and core-NafY have very weak interactions in intact NafY. The NMR structure of n-NafY reveals that it belongs to the sterile α-motif (SAM) family of domains, which are frequently involved in protein-protein interactions. The presence of a SAM domain in NafY was unexpected and could not be inferred from its amino acid sequence. Although SAM domains are very commonly found in eukaryotic proteins, they have rarely been identified in prokaryotes. The n-NafY SAM domain binds apo-NifDK. As opposed to full-length NafY, n-NafY impaired FeMo-co insertion when present in molar excess relative to FeMo-co and apo-NifDK. The implications of these observations are discussed to offer a plausible mechanism of FeMo-co insertion. NafY domain structure, molecular tumbling, and interdomain motion, as well as NafY interaction with apo-NifDK are consistent with the function of NafY in FeMo-co delivery to apo-NifDK.
The biological activation of N2 occurs at the FeMo‐cofactor, a 7Fe–9S–Mo–C–homocitrate cluster. FeMo‐cofactor formation involves assembly of a Fe6–8–SX–C core precursor, NifB‐co, which occurs on the ...NifB protein. Characterization of NifB‐co in NifB is complicated by the dynamic nature of the assembly process and the presence of a permanent 4Fe–4S cluster associated with the radical SAM chemistry for generating the central carbide. We have used the physiological carrier protein, NifX, which has been proposed to bind NifB‐co and deliver it to the NifEN protein, upon which FeMo‐cofactor assembly is ultimately completed. Preparation of NifX in a fully NifB‐co‐loaded form provided an opportunity for Mössbauer analysis of NifB‐co. The results indicate that NifB‐co is a diamagnetic (S=0) 8‐Fe cluster, containing two spectroscopically distinct Fe sites that appear in a 3:1 ratio. DFT analysis of the 57Fe electric hyperfine interactions deduced from the Mössbauer analysis suggests that NifB‐co is either a 4Fe2+–4Fe3+ or 6Fe2+–2Fe3+ cluster having valence‐delocalized states.
Des Clusters Kern: N2 wird in der Natur am FeMo‐Cofaktor aktiviert, einem 7Fe‐9S‐Mo‐C‐Homocitratcluster, der sich aus dem (6–8)Fe‐xS‐C‐Kern NifB‐co bildet, der am NifB‐Protein aufgebaut wird. Gemäß einer Mößbauer‐Analyse von in vivo aufgereinigtem NifB‐co im Komplex mit dem Carrierprotein NifX ist NifB‐co ein diamagnetischer (S=0) 8‐Fe‐Cluster mit zwei spektroskopisch unterschiedlichen Fe‐Zentren im Verhältnis 3:1.
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